The present invention relates to a laser microscope of the type that a target sample to be observed is irradiated with a pulse laser beam, particularly, to a multiphoton excitation scanning laser microscope for detecting the chemical reaction and fluorescent light caused by the multiphoton absorption of the sample.
In the multiphoton excitation method, the excitation which is performed in general by a single photon, is carried out by multiphoton. In, for example, a two-photon excitation method, the fluorescent light excitation, which is performed under a wavelength of 400 nm in the case of using a single photon, is carried out under a wavelength of 800 nm which is two times as long as the wavelength for the case of using a single photon.
Generally, in a mercury lamp or a continuous oscillation laser used in a fluorescent microscope, the photon density per unit time is low, leading to requirement of a tremendous light intensity for bringing about a multiphoton excitation phenomenon. Further, it is difficult to put the multiphoton excitation method to practical use unless the problem that a damage done to the optical system or to the sample is increased is solved.
Under the circumstances, a source, which can oscillate a pulse laser beam of, for example, sub-picosecond, is used as a light beam source of the multiphoton excitation method. This is because the multiphoton excitation phenomenon takes place in a probability which is substantially proportional to the square of the light density per unit area and unit time. In the pulse laser beam of a sub-picosecond, the probability of presence of a plurality of photons is increased.
For example, PCT National Publication No. 5-503149 discloses a two-photon excitation scanning laser microscope which employs a combination of a laser beam. source for emitting a pulse laser beam of sub-picosecond and a scanning optical unit for scanning the surface of a sample (the focal plane) with the pulse laser beam emitted from the laser beam source.
Pulse laser beams of sub-picosecond emitted from a laser beam source used for the multiphoton excitation do not have a wavelength of a complete single color, but has a wavelength range correlated with the pulse width. Generally, where light passes through an optical system, the speed of light within the medium is decreased with decrease in the wavelength and is increased with increase in the wavelength. It follows that, if a pulse laser beam having a wavelength range passes through an optical system, a difference in the passing time through the optical system is brought about, depending on different wavelengths. As a result, the pulse width after the passing through the optical system is expanded in the time axis direction, compared with the pulse width before the incidence on the optical system.
Since the probability of generation of the multiphoton excitation phenomenon is dependent on the photon density, the expansion of the pulse width on the sample surface or focal plane of the optical system lowers the probability of generation of the multiphoton excitation phenomenon. Accordingly, it is preferable to prevent the pulse width on the sample surface from expanding, as far as possible.
A so-called “pre-chirp compensation” is known as a general method of solving the above-noted problem. In the pre-chirp compensation, a pulse laser beam is passed through a prism pair or a grating pair so as to allow the light having a shorter wavelength to pass through the prisms or the gratings earlier than the light having a longer wavelength. In other words, the speed of light having a longer wavelength is retarded by the pre-chirp compensation.
The pre-chirp compensation is described in, for example, “Femtosecond pulse width control in microscopy by two-photon absorption autocorrelation; G. J. Brakenhoff, M. Muller & J. Squier; J. of Microscopy, Vol. 179, Pt. 3, September 1995, pp. 253-260. This literature teaches that the pulse width of a pulse laser beam can be optionally varied on a sample surface by controlling the position of the prism pair or the grating pair used as a pre-chirp compensator.
However, where the multiphoton excitation scanning laser microscope includes a plurality of objective lenses which are used selectively, the necessary degree of correction to be performed by the pre-chirp compensator is made different, depending on the object lenses, because the objective lenses differ from each other in the optical path length. Therefore, even if the pre-chirp compensator is adjusted to minimize the pulse width of the pulse laser beam on the sample surface on the basis of a single objective lens, the pulse width on the sample surface is increased in the case of another objective lens. Incidentally, the term “optical path length” used in this specification represents the optical length of a optical path formed by a optical element, not the geometrical length of the optical element.
In general, in a scanning laser microscope of this type, a target object in the sample is detected first by using an objective lens having a low magnification and capable of observation over a wide area, followed by using an objective lens of a high magnification for observation of fine portions, as in an ordinal general microscopic observation. If the pulse width of the pulse laser beam on the sample surface is changed by the switching of the objective lenses, it is impossible to cause the multiphoton excitation phenomenon under the optimum conditions.
If the difficulty is dealt with by the adjustment of a pre-chirp compensator, another problem is generated that the fluorescent light generated from the sample is faded in proportion to the time spent for the adjustment. As a countermeasure for preventing the fluorescent light of the sample from being faded, it is considered effective to move the sample from within the observation view field during the adjustment of the pre-chirp compensator. In this method, however, the sample must be moved again back into the observation view field after the adjustment of the pre-chirp compensator. What should be noted is that it is difficult to move the sample back exactly to the original position. It follows that this method is not practical for the observation and measurement.